Cyclosporin derivatives for treating inflammatory diseases and conditions

ABSTRACT

The present invention provides a method for the treatment of inflammatory diseases and/or conditions, e.g. allergic conjunctivitis, uveitis or phacoanaphylactic endophthalmitis in an eye of a mammal, said method comprising administering to said mammal in need of treatment a therapeutically effective amount of a novel cyclosporin A derivative selected from the group consisting of compounds represented by the formula: 
     
       
         
         
             
             
         
       
     
     wherein R 1  is S-Alk-R wherein Alk is an alkylene linkage, preferably a methylene or poly methylene linkage, or a polyalkenylene linkage, e.g. a C 3  to C 6  alkenylenyl linkage and R is a hydrogen or a unsubstituted or substituted hydrocarbyl group.

CROSS-REFERENCE

This is a continuation application of U.S. patent application Ser. No.12/785,133, filed on May 21, 2010 (now U.S. Pat. No. 8,524,671), whichclaims priority from U.S. Patent Application No. 61/181,382, filed onMay 27, 2009. The entire contents of all of the foregoing areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a method of treating inflammatory, includingocular and/or dermal, diseases and conditions having inflammation as acomponent of such ocular and/or dermal diseases and conditions, withcyclosporine derivatives. In particular, the present invention relatesto a method for the treatment of allergic conjunctivitis, aqueousdeficient dry-eye state, phacoanaphylaxis endophthalmitis and uveitisusing certain novel cyclosporine derivatives.

2. Description of the Related Art

Inflammation is the complex biological response of vascular tissues toharmful stimuli, such as pathogens, damaged cells, or irritants. It is aprotective attempt by the organism to remove the injurious stimuli aswell as initiate the healing process for the tissue.

In the absence of inflammation, wounds and infections would never healand progressive destruction of the tissue would compromise the survivalof the organism. However, inflammation which runs unchecked can alsolead to a host of diseases, such as hay fever, atherosclerosis, andrheumatoid arthritis. It is for this reason that inflammation isnormally tightly regulated by the body.

Inflammation can be classified as either acute or chronic. Acuteinflammation is the initial response of the body to harmful stimuli andis achieved by the increased movement of plasma and leukocytes from theblood into the injured tissues. A cascade of biochemical eventspropagates and matures the inflammatory response, involving the localvascular system, the immune system, and various cells within the injuredtissue. Prolonged inflammation, known as chronic inflammation, leads toa progressive shift in the type of cells which are present at the siteof inflammation and is characterized by simultaneous destruction andhealing of the tissue from the inflammatory process.

Acute inflammation is a short-term process which is characterized by theclassic signs of inflammation—swelling, redness, pain, heat, and loss offunction—due to the infiltration of the tissues by plasma andleukocytes. It occurs as long as the injurious stimulus is present andceases once the stimulus has been removed, broken down, or walled off byscarring.

The process of acute inflammation is initiated by the blood vesselslocal to the injured tissue, which alter to allow the exudation ofplasma proteins and leukocytes into the surrounding tissue. Theincreased flow of fluid into the tissue causes the characteristicswelling associated with inflammation since the lymphatic system doesn'thave the capacity to compensate for it, and the increased blood flow tothe area causes the reddened color and increased heat. The blood vesselsalso alter to permit the extravagation of leukocytes through theendothelium and basement membrane constituting the blood vessel. Once inthe tissue, the cells migrate along a chemotactic gradient to reach thesite of injury, where they can attempt to remove the stimulus and repairthe tissue.

Meanwhile, several biochemical cascade systems, consisting of chemicalsknown as plasma-derived inflammatory mediators, act in parallel topropagate and mature the inflammatory response. These include thecomplement system, coagulation system and fibrinolysis system.

Finally, down-regulation of the inflammatory response concludes acuteinflammation. Removal of the injurious stimuli halts the response of theinflammatory mechanisms, which require constant stimulation to propagatethe process. Additionally, many inflammatory mediators have short halflives and are quickly degraded in the tissue, helping to quickly ceasethe inflammatory response once the stimulus has been removed.

Chronic inflammation is a pathological condition characterized byconcurrent active inflammation, tissue destruction, and attempts atrepair. Chronic inflammation is not characterized by the classic signsof acute inflammation listed above. Instead, chronically inflamed tissueis characterized by the infiltration of mononuclear immune cells(monocytes, macrophages, lymphocytes, and plasma cells), tissuedestruction, and attempts at healing, which include angiogenesis andfibrosis.

Endogenous causes include persistent acute inflammation. Exogenouscauses are varied and include bacterial infection, prolonged exposure tochemical agents such as silica, tobacco smoke, or autoimmune reactionssuch as rheumatoid arthritis.

In acute inflammation, removal of the stimulus halts the recruitment ofmonocytes (which become macrophages under appropriate activation) intothe inflamed tissue, and existing macrophages exit the tissue vialymphatics. However in chronically inflamed tissue the stimulus ispersistent, and therefore recruitment of monocytes is maintained,existing macrophages are tethered in place, and proliferation ofmacrophages is stimulated.

The exudative component involves the movement of plasma fluid,containing important proteins such as fibrin and immunoglobulins(antibodies), into inflamed tissue. This movement is achieved by thechemically-induced dilation and increased permeability of blood vessels,which results in a net loss of blood plasma. The increased collection offluid into the tissue causes edema.

Acute inflammation is characterised by marked vascular changes,including vasodilation, increased permeability, and the slowing of bloodflow, which are induced by the actions of various inflammatorymediators. Vasodilation occurs first at the arteriole level, progressingto the capillary level, and brings about a net increase in the amount ofblood present, causing the redness and heat of inflammation. Increasedpermeability of the vessels results in the movement of plasma into thetissues, with resultant stasis due to the increase in the concentrationof the cells within blood—a condition characterized by enlarged vesselspacked with cells. Stasis allows leukocytes to marginate along theendothelium, a process critical to their recruitment into the tissues.Normal flowing blood prevents this, as the shearing force along theperiphery of the vessels moves cells in the blood into the middle of thevessel.

Abnormalities associated with inflammation comprise a large, unrelatedgroup of disorders which underly a variety of human diseases. The immunesystem is often involved with inflammatory disorders, demonstrated inboth allergic reactions and some myopathies, with many immune systemdisorders resulting in abnormal inflammation. Non-immune diseases withaetiological origins in inflammatory processes are thought to includecancer, atherosclerosis, and ischemic heart disease.

A large variety of proteins are involved in inflammation, and any one ofthem is open to a genetic mutation which impairs or otherwisederegulates the normal function and expression of that protein.

Examples of disorders associated with inflammation include:

-   -   Asthma    -   Autoimmune diseases    -   Chronic inflammation    -   Chronic prostatitis    -   Glomerulonephritis    -   Hypersensitivities    -   Inflammatory bowel diseases    -   Pelvic inflammatory disease    -   Reperfusion injury    -   Rheumatoid arthritis    -   Transplant rejection    -   Vasculitis

The inflammatory response must be actively terminated when no longerneeded to prevent unnecessary damage to tissues. Failure to do soresults in chronic inflammation, cellular destruction, and attempts toheal the inflamed tissue. One intrinsic mechanism employed to terminateinflammation is the short half-life of inflammatory mediators in vivo.They have a limited time frame to affect their target before breakingdown into non-functional components, therefore constant inflammatorystimulation is needed to propagate their effects.

Active mechanisms which serve to terminate inflammation include

-   -   TGF-β from macrophages    -   Anti-inflammatory lipoxins    -   Inhibition of pro-inflammatory molecules, such as leukotrienes

Specific diseases and conditions of the eye having an inflammatorycomponent include allergic conjunctivitis, phacoanaphylacticendophthalmitis and uveitis. These diseases and conditions can belocated throughout the eye, in both the posterior and anterior chambersof the eye as well as in the vitreous body.

Uveitis, the inflammation of the uvea, is responsible for about 10% ofthe visual impairment in the United States. Phacoanaphylacticendophthalmitis is a human autoimmune disease.

Panuveitis refers to inflammation of the entire uveal (vascular) layerof the eye. Posterior uveitis generally refers to chorioentinitis, andanterior uveitis refers to iridocyclitis. The inflammatory products(i.e. cells, fibrins, excess proteins) of these inflammations arecommonly found in the fluid spaces if the eye, i.e. anterior chamber,posterior chamber and vitreous space as well as infiltrating the tissueintimately involved in the inflammatory response. Uveitis may occurfollowing surgical or traumatic injury to the eye; as a component of anautoimmune disorder, i.e. rheumatoid arthritis, Behcet's disease,ankylosing spondylitis, sarcoidosis; as an isolated immune mediatedocular disorder, i.e. pars planitis, iridocyclitis etc., unassociatedwith known etiologies; and following certain systemic diseases whichcause antibody-antigen complexes to be deposited in the uveal tissues.Together these disorders represent the non-infectious uveitities.

Cyclosporins have been used to treat inflammatory conditions includingan autoimmune component such as arthritis (for example rheumatoidarthritis, arthritis chronica progrediente and arthritis deformans) andrheumatic diseases. Specific auto-immune diseases for which Cyclosporinhas been proposed or applied include, autoimmune hematological disorder(including e.g. hemolytic anaemia, aplastic anaemia, pure red cellanaemia and idiopathic thrombocytopaenia), systemic lupus erythematosus,poly-chondritis, sclerodoma, Wegener granulamatosis, dermatomyositis,chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnsonsyndrome, idiopathic sprue, autoimmune inflammatory bowel disease(including e.g. ulcerative colitis and Crohn's disease) endocrineopthalmopathy, Graves disease, sarcoidosis, multiple sclerosis, primarybiliary cirrhosis, juvenile diabetes (diabetes mellitus type 1), uveitis(anterior and posterior), keratoconjunctivitis sicca and vernalkeratoconjunctivitis, interstitial lung fibrosis, psoriatic arthiritsand glomerulonephritis (with and without nephrotic syndrome, e.g.including idiopathic nephrotic syndrome or minimal change nephropathy).(See U.S. Pat. No. 6,346,511.)

Thus, it will be understood that it is desirable to develop compoundsthat are useful in treating inflammatory diseases and conditions. It hasbeen found that the compounds disclosed herein may be used to treatvarious inflammatory diseases and conditions.

Such compounds include the methylthio-substituted cyclosporin A andother alkylthio-substituted cyclosporin A derivatives described in PCTapplication Nos. 98-379455, 98-379456 and 98-379457, which have beenfound to be active against certain retroviruses, especially AIDS(acquired immunodeficiency syndrome) and ARC (AIDS-related complex) whenadministered orally, parenterally, rectally or by inhalation. Inaddition, these compounds have generally been found to have only a veryweak immunosuppressant action, and to show anti-retroviral activity atnon-cytotoxic and non-cytostatic concentrations. These compounds areclaimed to have a synergistic action with other agents active againstretrovirus (such as inhibitors of reverse transcriptase, protease,integrase, HIV replication and nucleocapside). (See also U.S. Pat. Nos.5,944,299; 5,977,067; 5,965,527 and 5,948,755)

These compounds are also claimed for use in the treatment of oculardiseases and conditions in U.S. Pat. Nos. 6,350,442 and 6,254,860.

Thus, it is one object of this invention to treat inflammatory diseasesand conditions, including diseases and conditions having inflammation asa component thereof, with cyclosporine derivatives.

It is another object of this invention to provide cyclosporine Aderivatives to treat ocular diseases and conditions, such as allergicconjunctivitus.

It is one object of this invention to treat ocular diseases andconditions, including ocular diseases and conditions having inflammationas a component thereof, with cyclosporine derivatives.

It is another object of the invention to treat dermal diseases andconditions, including dermal diseases and conditions having inflammationas a component thereof, with cyclosporine derivatives.

It is another object of the invention to treat dermal conditions, suchas psoriasis and dermatitis.

Other objects of this invention will become apparent from a reading ofthe present specification.

SUMMARY OF THE INVENTION

The present invention provides a method for treating an inflammatorydisease, disorder or condition of a mammal, e.g. a human, such as aninflammatory disease, disorder or condition of the eye, for example,allergic conjunctivitis, uveitis, or phacoanaphylactic endophthalmitis,comprising the step of administering to a patient in need thereof,including topically or systemically administering to the eye of suchpatient, a therapeutically effective amount of a compound selected fromthe group consisting of cyclosporin A derivatives of the formuladescribed below. The present invention preferably provides a method fortreating a disorder or condition of the eye, with a therapeuticallyeffective amount of a compound selected from the group consisting ofcyclosporin A derivatives. The cyclosporin A derivatives utilized in themethod(s) of the present invention are represented by the formula

wherein R₁ is S-Alk-R wherein Alk is an alkylene linkage, preferably amethylene or poly methylene linkage, e.g. a C₂ to C₆ polymethylenelinkage, or a polyalkenylene linkage, e.g. a C₃ to C₆ alkenylenyllinkage, and R is hydrogen or a unsubstituted or substituted hydrocarbylgroup. Preferably, R is a nitrogen-containing hydrocarbyl group, e.g. apoly nitrogen-containing hydrocarbyl group, having 2 or 3 nitrogenatoms, i.e. an amidine or guanidine-containing hydrocarbyl radical. Inparticular, R may be selected from the group consisting of radicals ofthe following formulae: R is —N═C(NR₃R₄)(NR₅R₆) or

—NR₇[(NR₃R₄)C═NR₅], i.e. guanidines or —N═C(R₈)(NR₉R₁₀), i.e. amidines,wherein R₃-R₁₀ is H, Alk, Ar or (CH₂)nAr wherein Ar is an aryl group andn is an integer of from 1 to 13 or R₃ and R₄, or R₄ and R₅, or R₅ andR₇, or R₃ and R₇, or R₉ and R₁₀, or R₈ and R₉, together, may be—(CH₂)_(x)—, wherein x is an integer of from 2 to 5, e.g. CH₂—CH₂— or—CH₂—CH₂—CH₂—.

R₂ may be selected from the group consisting of hydroxyl, lower alkyland hydroxyl-substituted lower alkyl.

For example, R₁ may be —S(CH₂)₂N═C(NH₂)₂ and R₂ may be —CH₂CH(CH₃)₂,

—CH₂C(OH)(CH₃)₂, —CH(CH₃)₂ or —CH(CH₃)CH₂CH₃.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the treatment ofinflammatory diseases and conditions, including diseases and conditionsof the eye, having an inflammatory component associated therewith, e.g.by topical application to the affected eye, of a cyclosporin derivative,represented by the formula below

wherein R₁ and R₂ are defined above. In particular R₁ is S-Alk-R whereinAlk is an alkylene linkage, preferably a methylene or poly methylenelinkage, e.g. a C₂ to C₆ polymethylene linkage, or a polyalkenylenelinkage, e.g. a C₃ to C₆ alkenylenyl linkage and R₂ is selected from thegroup consisting of hydroxyl, lower alkyl and hydroxyl substituted loweralkyl.

In a first aspect of the invention, R is —N═C(NR₃R₄)(NR₅R₆) or—NR₇[(NR₃R₄)C═NR₅], i.e. a guanidine or —N═C(R₈)(NR₉R₁₀), i.e. anamidine wherein R₃-R₁₀ is H, Alk, Ar or (CH₂)nAr wherein Ar is an arylgroup and n is an integer of from 1 to 13 or R₃ and R₄, or R₄ and R₅, orR₅ and R₇, or R₃ and R₇, or R₉ and R₁₀, or R₈ and R₉, together, may be—(CH₂)_(x)—, wherein x is an integer of from 2 to 5, e.g. —CH₂—CH₂— or—CH₂—CH₂—CH₂—.

In a second aspect of the invention, R₁ is a hydrogen atom or a radicalof formula (Ia):

—S-Alk-R₁₁  (Ia)

in which

Alk-R₁₁ represents a methyl radical, or alternatively

Alk represents a C₂-C₆ straight chain or branched alkylene radical or aC₃-C₆ cycloalkylene radical, and

R₁₁. represents

a hydrogen atom or a hydroxyl, carboxyl or alkyloxycarbonyl radical, or

an —NR₁₂R₁₃ radical in which R₁₂ and R₁₃, which are identical ordifferent, represent a hydrogen atom or a phenyl, alkyl, C₂-C₄ alkenylor C₃-C₆ cycloalkyl radical, said radical optionally substituted withselected from a halogen atom, an alkyloxy, alkyloxycarbonyl, amino,alkylamino and dialkylamino radical; or

R₁₂ and R₁₃ represent a benzyl or saturated or unsaturated heterocycylicradical, said heterocycylic radical containing from 5 to 6 ring membersand from 1 to 3 heteroatoms;

or in which R₁₂ and R₁₃ form, together with the nitrogen atom to whichthey are attached, a saturated or unsaturated 4- to 6-memberedheterocycle, which heterocycle having an additional heteroatom selectedfrom nitrogen, oxygen and sulphur, and wherein said saturated orunsaturated heterocycle is optionally substituted by an alkyl, phenyl orbenzyl radical, or R₁ is a radical of the formula (Ib):—N(R₁₄)—(CH₂)_(n)—NR₁₂R₁₃ in which R₁₂ and R₁₃ are as defined above, R₁₄represents a hydrogen atom or an alkyl radical and n is an integerranging from 2 to 4,

and R₂ is selected from the group consisting of hydroxyl, lower alkyland hydroxyl substituted lower alkyl,

with the proviso that, when R₁ is a hydrogen atom, then R₂ is not analkyl butyl? radical, and wherein the alkyl portions or radicals definedabove are straight chain or branched and contain from 1 to 4 carbonatoms, or a pharmaceutically acceptable salt thereof.

In the cyclosporine A derivatives of this second aspect of theinvention, the trans butene moiety, which is normally present in the1-position of cyclosporine A, may be replaced with R₁₅ wherein R₁₅represents a radical of formula

—CH₂CHCHCH₂—R₁₆ (Ic) or —CH₂SR₁₇ (Id), wherein R₁₆ represents analkylthio, aminoalkylthio, alkylaminoalkylthio, dialkylaminoalkylthio,pyrimidinylthio, thiazolylthio, N-alkylimidazolylthio,hydroxyalkylphenylthio, hydroxyalkylphenyloxy, nitrophenylamino or2-oxopyrimidin-1-yl radical and R₁₇ represents an alkyl radical.

This invention also provides pharmaceutical compositions for topicalapplication in the treatment of an inflammatory disease, disorder orcondition of a mammal, e.g. a human, such as an inflammatory disease,disorder or condition of the eye, for example, allergic conjunctivitis,uveitis, or phacoanaphylactic endophthalmitis, comprising the step ofadministering to a patient in need thereof, including topically orsystemically administering to the eye of such patient, a therapeuticallyeffective amount of a compound selected from the group consisting of theabove cyclosporin A derivatives.

For the purpose of describing and claiming the present invention thefollowing terms shall have the following meanings:

“Alkyl” refers to a straight-chain, branched or cyclic saturatedaliphatic hydrocarbon. Preferably, the alkyl group has 1 to 12 carbons.More preferably, it is a lower alkyl of from 1 to 7 carbons, mostpreferably 1 to 4 carbons. Typical alkyl groups include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl andthe like. The alkyl group may be optionally substituted with one or moresubstituents are selected from the group consisting of hydroxyl, cyano,alkoxy, ═O, ═S, NO₂, halogen, dimethyl amino and SH.

“Alkenyl” refers to a straight-chain, branched or cyclic unsaturatedhydrocarbon group containing at least one carbon—carbon double bond.Preferably, the alkenyl group has 2 to 12 carbons. More preferably it isa lower alkenyl of from 2 to 7 carbons, most preferably 2 to 4 carbons.The alkenyl group may be optionally substituted with one or moresubstituents selected from the group consisting of hydroxyl, cyano,alkoxy, O, S, NO₂, halogen, dimethyl amino and SH.

“Aryl” refers to an aromatic group which has at least one ring having aconjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups. The aryl group may be optionallysubstituted with one or more substituents selected from the groupconsisting of halogen, trihalomethyl, hydroxyl, SH, OH, NO₂, amine,thioether, cyano, alkoxy, alkyl, and amino.

“Alkaryl” refers to an alkyl that is covalently joined to an aryl group.Preferably, the alkyl is a lower alkyl.

“Alkoxy” refers to an “O-alkyl” group.

“tBoc” refers to a t-butyloxycarbonyl protecting group.

“Carbocyclic aryl” refers to an aryl group wherein the ring atoms arecarbon.

“Heterocyclic aryl” refers to an aryl group having from 1 to 3heteroatoms as ring atoms, the remainder of the ring atoms being carbon.Heteroatoms include oxygen, sulfur, and nitrogen.

The cyclosporine A derivatives used in the method of this invention areprepared as follows:

Compounds where R₄, R₅ and R₆ are hydrogen and R₇ is hydrogen, alkyl,substituted alkyl or aryl may be prepared by reaction of a compound offormula (I) where X is a leaving group and P is a protecting group witha compound of formula (II) in a suitable solvent such as methanol toafford compounds of formula (III). For compounds of formula I, typicalexamples of the protecting group are where X=chlorine, MeS, MeSO2,1-imidazolyl and especially 1-pyrazolyl.

Protecting groups P are preferably tertiary butyloxycarbonyl groups(tBoc) groups.

Compounds of formula (III) may be de-protected under a variety ofconditions to provide compounds of formula (IV). For example, whenP=tertiary butyloxycarbonyl groups (tBoc), this may be removed underacidic conditions using acids such as methanesulphonic acid.

Compounds of formula (V) where R₇ is hydrogen, alkyl, substituted alkylor aryl; R₃ is alkyl, substituted alkyl or aryl, may be prepared byreaction of a compound of formula (VI) where X is a leaving group and Pis a protecting group with a compound of formula (II) in a suitablesolvent such as methanol to afford compounds of formula (VII).

For compounds of formula (VI), typical examples of the protecting groupare where X=chlorine, MeS, MeSO2, 1-imidazolyl and especially1-pyrazolyl. Protecting groups P are preferably tertiarybutyloxycarbonyl groups (tBoc) groups.

For example, inWO/2003/051797N,N′-Di-tBoc-N-methyl-1H-pyrazole-1-carboxamidine has beenused to prepare an N-methyl guanidine in an unrelated chemical family.

Other compounds of the invention may be made in similar ways usingrelated synthetic methods with, if appropriate, suitable protectinggroups compatible with the synthetic methodology.

Compounds of formula (X) where R is —N═C(R₈)—NR₉R₁₀ (amidines) where R₈is hydrogen, alkyl, substituted alkyl or aryl and R₉ and R₁₀ can bealkyl, substituted alkyl or aryl or R₉ and R₁₀ can form a ring may beprepared by reaction of a compound of formula (VIII) with a compound offormula (IX) to afford compounds of formula (X).

R₁₁ is preferably lower alkyl and typical examples of compound (VIII)are Dimethylformamide dimethylacetal (DMF.DMA) and Dimethylacetamidedimethylacetal (DMA.DMA).

Below are specific examples of the preparation of certain compounds ofthe invention by the above general procedures.

Guanidine and Amidine Analogues of 3-[(2-aminoethylthio]-cyclosporin A

Example A 3-[(2-Guanidyl)-ethylthio]-cyclosporin A (III)

To a solution of 3-[(2-aminoethylthio]-cyclosporin A*-(I)) (200 mg, 0.16mmol) in methanol (20 mL) was added di-tBoc-pyrazole carboxamidine (250mg, 0.8 mmol), and the reagents were stirred together for 18 h. Afterthis time, a further portion of the di-Tt-pyrazole carboxamidine (100mg, 0.32 mmol) was added and the reaction was stirred for a further 3 h.The reaction was then reduced in vacuo, redissolved in dichloromethane,washed with 0.5M citric acid, and the organic layer was dried over MgSO₄and reduced in vacuo. The product was then purified by chromatographycolumn on a 10 g SPE cartridge eluting with diethyl ether to isolate 90mg (40%) of desired product (II).

As the first member of the Guanidine and Amidine examples synthesizedand because of the difficulties anticipated in characterising the finalproduct (III), it was decided to fully and extensively characterize thedi-tBoc protected guanidine (II) at this stage and to then take thismaterial onto the free guanidine (III) by acid hydrolysis. Subsequentanalogues in this Guanidine and Amidine subclass made from3-[(2-aminoethylthio]-cyclosporin A were then characterised principallyby MS

Compound (II) was analysed by ¹H, ¹³C, DEPT NMR and subsequently by aseries of 2-D NMR experiments, HMQC, HMBC and DEPT-HMQC.

Presence of the 3-[(2-Guanidyl)-ethylthio]side chain was confirmed by 1D& 2D NMR.

Analysis was performed in CDCl₃ solution at 300K on a Bruker DRX500spectrometer.

¹H NMR Key Resonances:

δ=1.50, 1.51 ppm (2 singlets, 2×Boc, 18H, 6×CH3)δ=5.89 ppm, (singlet, sarcosine, 1H)

2D Spectra

Using ¹H detected Hetronuclear Multiple Quantum Coherence (HMQC),Hetronuclear Multiple Bond Correlation (HMBC) and edited HetronuclearSingle Quantum Coherence (DEPT-HSQC) experiments, connectivity andassignment may be made confirming the presence of the3-[(2-Guanidyl)-ethylthio]side chain.

H (3) to 2′ (multiplet, ¹H 2.84 ppm, 2H).2′ to 3′ (multiplet, ¹H 3.67 ppm, 2H).3′ to NH 4′ (triplet J_(HH) 5.8 Hz, 1H 8.67 ppm, 1H).

To a solution of the di-tBoc protected3-[(2-Guanidyl)-ethylthio]-cyclosporin A (II) (21 mg, 0.0138 mmol) indichloromethane (0.3 mL) was added trifluoroacetic acid (0.3 mL) and thesolution was stirred at room temperature for 1 hour. The solution wasconcentrated to give the product (III) as a white solid (20 mg; 100%)

Analysis by MS (E⁺) showed a mass of 1320.2 (M+H) consistent with theproposed structure

Example B 3-[(2-N,N-dimethylformamidinyl)-1-thioethyl]-cyclosporin A(III)

A mixed solution of 3-(1-thioethylamine)cyclosporine A (0.64 g, 0.5mmol) and N,N-dimethylformamide dimethyl acetal in 20 mL of THF wasrefluxed for two hours. After removal of solvent under vacuum, theresidue was subject to silica gel column using methylene/methanol (10:1)as eluents, 300 mg of pure product was obtained (yield: 45.0%)

MS (E+) showed a mass of 1332.82 (M+H⁺) consistent with the proposedstructure.

Other methods will be apparent to a chemist skilled in the art as willmethods for preparing starting materials and intermediates etc

In accordance with the present invention, the cyclosporin A derivativesmay be applied, e.g. to an affected eye, in any efficaciousconcentration. When said cyclosporin A derivatives are applied to theeye as a topical ophthalmic composition the composition may comprise,0.01 to saturation (e.g. greater than 20 weight percent) of saidcyclosporin A derivative in a pharmaceutically acceptable excipient.From 0.01 to 50 weight percent, preferably from 0.1 to 20 weightpercent, of cyclosporin A derivatives in a pharmaceutically acceptableexcipient may be used. Such pharmaceutically acceptable excipients are,for example, animal oil, vegetable oil, an appropriate organic oraqueous solvent, an artificial tear solution, a natural or syntheticpolymer, or an appropriate membrane to encapsulate the cyclosporin Aderivative.

Specific examples of these pharmaceutically acceptable excipients areolive oil, arachis oil, castor oil, mineral oil, petroleum jelly,dimethyl sulphoxide, chremophor, Miglyol 182 (commercially availablefrom Dynamit Nobel Kay-Fries Chemical Company, Mont Vale, N.J.), analcohol (e.g. ethanol, n-propyl alcohol, or iso-propyl alcohol),liposomes or liposome-like products or a silicone fluid. Preferredexcipients are dimethyl sulphoxide and olive oil. Mixtures of at leasttwo of any suitable excipients may be used.

Examples of artificial tear excipients which can be advantageously usedin the practice of this invention are isotonic sodium chloride,cellulose ethers such as hydroxypropylmethylcellulose andhydroxyethylcellulose, polyvinyl alcohol and available artificial teasolutions.

An example of a useful polymeric excipient is a polyoxyethylated castoroil.

Examples of pharmaceutically acceptable membranes which can beadvantageously used in the practice of this invention are microdone, anartificial lipid membrane, polyvinyl alcohol, or methylcellulose.

The cyclosporin A derivatives are advantageously administered topicallyas an ophthalmic drop (solution or suspension) or ophthalmic ointmentcontaining an effective amount of the derivative. Concentrations of 0.01to 50 weight percent, preferably 0.1 to 20 weight percent, of thecyclosporin A derivatives are used in the practice of the presentinvention.

In accordance with a method of the present invention, at least one ofthe cyclosporin A derivatives is administered topically in any quantityrequired to provide the degree of treatment needed. For example, 5microliters to 1 milliliter of a solution, suspension, or ointmentcontaining an effective amount of the cyclosporin A derivative, such as0.01 to 50 weight percent, preferably 0.1 to 20 weight percent, of thecyclosporin A derivative is advantageously used.

Numerous advantages accrue with the practice of the present invention.The method of the present invention is useful in that it can locallyprevent activation of a presystemic response.

Topical administration of the cyclosporin A derivatives to a patient'stear deficient eye increases tear production in the eye. Thus, suchtreatment further serves to correct corneal and conjunctival disordersexacerbated by tear deficiency and KCS, such as corneal scarring,corneal ulceration, inflammation of the cornea or conjunctiva,filamentary keratisis, mucopurulent discharge and vascularization of thecornea. Furthermore, the cyclosporin A derivatives directly decrease theimmune response and granulation and neovascularization.

Example 1

This example compares the relative potency of the cyclosporine analoguesutilized in the method of the present invention with cyclosporine A inmodulating Jurkat T cell functions.

A. Inhibition of T Cell Proliferation.

In this experiment the % Viable cells @ 10 uM for 48 hours is determinedby WST Assay (Water Soluble Tetrazolium).

Compound % Viable cells Cyclosporin A 57 CsA-A1 45 CsA-A2 62 CsA-A3 72CsA-A4 65 CsA-A5 78 CsA-A6 94

The results of this experiment shows that the cyclosporine analoguesutilized in the method of the present invention are as good or betterthan cyclosporine A.

B. Induction of T cell apoptosis

The percentage (%) of apoptic T cells is determined @ 10 uM compoundconcentration for 24 hours of incubation period.

Cyclosporin A 51 CsA-A1 62 CsA-A2 31 CsA-A3 18 CsA-A4 34 CsA-A5 25CsA-A6 14

Again, the compound CsA-A1 shows surprisingly greater effect thancyclosporine A.

C. Suppression of IL-2 Production by T Cells

The percentage (%) inhibition in IL-2 production is determined 10 uMcompound concentration for 24 hours of incubation period.

Cyclosporin A 100 CsA-A 1 100 CsA-A2 82 CsA-A3 28 CsA-A4 19 CsA-A5 36CsA-A6 4

The compound CsA-A1, surprisingly, shows equivalent effectiveness ascompared to cyclosporine A in this experiment.

As a result of the experiments described in Example 1, CsA-A1 wasselected for further work.

Example 2

This example compares the relative potency of CsA-A1, the most preferredcyclosporine A derivative utilized in the method of the presentinvention with cyclosporine A in an in-vivo modal of ocularinflammation.

A. Rat EIU Model: Acute Intraocular Inflammation

Rats were injected in the foot pad LPS (100 ug/rat.) 5 rats were treatedin each of the following groups. The topical vehicle was the Restasisvehicle.

The systemic vehicle was:

Ethanol (200 Proof) 15% Cremaphor EL 15% Polysorbate 80  2% Water 68%

Topically (t.i.d.) Systemically (i.p., b.i.d) Vehicle 25 mg/kgCyclosporin A (ethanol) 0.05% Cyclosporin 25/mg/kg CsA-A1(ethanol) 0.05%CsA-A1

Samples of the blood and aqueous humor were collected and analyzed 24hours post LPS injection. The histology of the cells was also recordedThe results are shown in FIG. 1.A. As can be observed in FIGS. 1 Athrough E CsA-A1 showed a similar potency to cyclosporine A ininhibiting acute intraocular inflammation when applied topically and/orsystemically in this experiment.

This example compares the relative potency of CsA-A1, the most preferredcyclosporine A derivative utilized in the method of the presentinvention with cyclosporine A in an in-vivo modal of ocularinflammation.

B. Rat EAU Model: Chronic Intraocular Inflammation Rats were immunizedwith R16 (RIBP immunogenic peptide, 100 ug/rat. The rats were thenfitted with Alzet Pump to deliver 5 mg/kg/day of the test sample, below,or injected intraperitoneal with 10 mg/kg, q.d. of the test sample. 5rats were treated in each of the following groups. The vehicle was

Ethanol (200 Proof) 15% Cremaphor EL 15% Polysorbate 80  2% Water 68%

In vivo fluorophotometry was performed on days 9, 12 and 14 postimmunization. Aqueous samples and globes were collected and analyzed onday 14.

The results are shown in FIG. 2. s can be observed in FIGS. 2 A through2K E CsA-A1 showed a similar potency to cyclosporine A in inhibitingacute intraocular inflammation when applied by intraperitoneal injectionin this experiment. However, as a result of its lower solubility in thevehicle, it appeared to be less effective when applied by the AlzetPump.

The Rat EAU model is indicative of autoimmune-mediated inflammatoryconditions. Indeed, uveitis may precede, accompany or develop followingthe onset of many systemic autoimmune diseases including juvenilerheumatoid arthritis (RA), psoriatic arthritis, multiple sclerosis (MS),inflammatory bowel disease and systemic lupus erythematosus. Theetiology of these chronic inflammatory conditions is not wellunderstood; however, evidence suggests that autoreactive T cells andantibodies recognizing self-antigens localized within affected tissuesdrives the chronic inflammatory response, e.g. uveal and/or retinalantigens, myelin proteins within central nervous system or components ofcartilage in patients with uveitis, MS and RA respectively. In any case,environmental factors (e.g. viral or bacterial infection) in the contextof a genetic predisposition (e.g. mutations in genes encoding HLA) arethought to break self-tolerance and trigger the onset of disease. Thisconcept is exemplified in EAU and other models of autoimmune-mediateddisease. Lewis rats immunized with uveal or retinal antigens, includingS-antigen or interphotoreceptor retinoid binding protein (IRBP), exhibitsimilar clinical and histological disease profiles compared to patientswith posterior uveitis. Genetically susceptible strains of miceimmunized with type II collagen show histopathological and clinicalsimilarities to patients with rheumatoid arthritis. Likewise,immunization with myelin peptides mimics the immunpathogenesis of MS,including myelin loss within white matter tracts of the CNS andparalysis, and these mice may also develop uveitis.

Further objects of this invention, together with additional featurescontributing thereto and advantages accruing therefrom will be apparentfrom the following examples of the invention.

The foregoing description details specific methods and compositions thatcan be employed to practice the present invention, and represents thebest mode contemplated. Thus, however detailed the foregoing may appearin text, it should not be construed as limiting the overall scopehereof; rather, the ambit of the present invention was to be governedonly by the lawful construction of the appended claims. In particular,although the method of the present invention has been described with theuse of the specific cyclosporine A derivatives of the above formula, thenovel cyclosporine derivatives that may be used in the method of thepresent invention further include 3-substituted iminoalkylthiocyclosporin A derivatives, preferably 3-substituteddiaminoiminoalkylthio cyclosporin A derivatives, e.g.((R)-(diamino)iminoalkyllthio-Sar)³-(4′-hydroxy-MeLeu)⁴ cyclosporin A,((R)-(alkyl)(dialkylamino)iminoalkylthio-Sar)³-(4′-hydroxy-MeLeu)⁴-cyclosporinA, ((R)-(alkyl)(dialkylamino)iminoalkylthio-Sar)³-cyclosporin Aderivatives and ((R)-(diamino)iminoalkylthio-Sar)³-cyclosporin Aderivatives.

The invention may be summarized as follows.

1. A method for the treatment of an inflammatory disease, disorder orcondition of a mammal, comprising the step of administering to a patientin need thereof, a therapeutically effective amount of a cyclosporin Aderivative selected from the group consisting of compounds representedby the formula:

wherein R₁ is S-Alk-R wherein Alk is an alkylene linkage and R is ahydrogen or a unsubstituted or substituted hydrocarbyl group.

2. The method of 1 wherein Alk is a methylene or a C₃ to C₆ alkenylenyllinkage.

3. The method of 1 wherein R₁ is a methylene or a C₂ to C₆ polymethylenelinkage.

4. The method of 1 wherein R₁ is a C₃ to C₆ alkenylenyl linkage.

5. The method of 1 wherein R is —N═C(NR₃R₄)(NR₅R₆) or —NR₇C(NR₃)(C═NR₅),wherein R₃-R₇ is H, Alk, Ar or (CH₂)nAr wherein Ar is an aryl group andn is an integer of from 1 to 13 or R₃ and R₄ or R₄ and R₅ or R₅ and R₇or R₃ and R₇, together may be —CH₂—CH₂— or —CH₂—CH₂—CH₂—.

6. The method of 1 wherein said cyclosporine A derivative is a3-substituted diaminoiminoalkylthio cyclosporine A derivative.

7. The method of 1 wherein said cyclosporine A derivative is selectedfrom the group consisting of((R)-(diamino)iminoalkyllthio-Sar)³-(4′-hydroxy-MeLeu)⁴ cyclosporin Aderivatives,((R)-(alkyl)(dialkylamino)iminoalkylthio-Sar)³-(4′-hydroxy-MeLeu)⁴-cyclosporinA, ((R)-(alkyl)(dialkylamino)iminoalkylthio-Sar)³-cyclosporin Aderivatives and ((R)-(diamino)iminoalkylthio-Sar)³-cyclosporin Aderivatives.

8. The method of 1 wherein said cyclosporine A derivative is selectedfrom the group of compounds according to 1 wherein R₁ is—S(CH₂)₂N═C(NH₂)₂ and R₂ is —CH₂CH(CH₃)₂, —CH₂C(OH)(CH₃)₂, —CH(CH₃)₂ or—CH(CH₃)CH₂CH₃.

9. The method of 1 wherein the compound is administered in a compositioncomprising 0.1 to 20 wt % of the compound together with apharmaceutically acceptable excipient.

10. The method of 9 wherein the pharmaceutically acceptable excipient isselected from the group consisting of animal oil and vegetable oil.

11. The method of 9 wherein the pharmaceutically acceptable excipient isselected from the group consisting of olive oil, arachis oil, castoroil, mineral oil, petroleum jelly, dimethyl sulphoxide, an alcohol,silicone fluid and mixtures thereof.

12. The method of 1 wherein R₁ is a hydrogen atom or a radical offormula (Ia):

—S-Alk-R₁  (Ia)

in which

Alk-R₁₁ represents a methyl radical, or alternatively

Alk represents a C₂-C₆ straight chain or branched alkylene radical or aC₃-C₆ cycloalkylene radical, and

R₁₁. represents

a hydrogen atom or a hydroxyl, carboxyl or alkyloxycarbonyl radical, or

an —NR₁₂R₁₃ radical in which R₁₂ and R₁₃, which are identical ordifferent, represent a hydrogen atom or a phenyl, alkyl, C₂-C₄ alkenylor C₃-C₆ cycloalkyl radical, said radical optionally substituted withselected from a halogen atom, an alkyloxy, alkyloxycarbonyl, amino,alkylamino and dialkylamino radical; or

R₁₂ and R₁₃ represent a benzyl or saturated or unsaturated heterocycylicradical, said heterocycylic radical containing from 5 to 6 ring membersand from 1 to 3 heteroatoms;

or in which R₁₂ and R₁₃ form, together with the nitrogen atom to whichthey are attached, a saturated or unsaturated 4- to 6-memberedheterocycle, which heterocycle having an additional heteroatom selectedfrom nitrogen, oxygen and sulphur, and wherein said saturated orunsaturated heterocycle is optionally substituted by an alkyl, phenyl orbenzyl radical, or R₁ is a radical of the formula (Ib):—N(R₁₄)—(CH₂)_(n)—NR₁₂R₁₃ in which R₁₂ and R₁₃ are as defined above, R₁₄represents a hydrogen atom or an alkyl radical and n is an integerranging from 2 to 4,

and R₂ is selected from the group consisting of hydroxyl, lower alkyland hydroxyl substituted lower alkyl,

with the proviso that, when R₁ is a hydrogen atom, then R₂ is not analkyl butyl? radical, and wherein the alkyl portions or radicals definedabove are straight chain or branched and contain from 1 to 4 carbonatoms, or a pharmaceutically acceptable salt thereof.

13. The method of 12 wherein in the cyclosporine A derivatives of theformula, the trans butene moiety, which is normally present in the1-position of cyclosporine A, is replaced with R₁₅ wherein R₁₅represents a radical of formula

—CH₂CHCHCH₂—R₁₆ (Ic) or —CH₂SR₁₇ (Id), wherein R₁₆ represents analkylthio, aminoalkylthio, alkylaminoalkylthio, dialkylaminoalkylthio,pyrimidinylthio, thiazolylthio, N-alkylimidazolylthio,hydroxyalkylphenylthio, hydroxyalkylphenyloxy, nitrophenylamino or2-oxopyrimidin-1-yl radical and R₁₇ represents an alkyl radical.

14. The method of 12 wherein R represents

a hydroxyl radical,

a carboxyl radical,

an alkyloxycarbonyl radical,

an —NR₁₂R₁₃ radical in which R₁₂ and R₁₃, which are identical ordifferent, represent a hydrogen atom or an alkyl, C₂-C₄ alkenyl, C₃-C₆cycloalkyl or optionally substituted phenyl radical, wherein said phenylradical may be substituted by a halogen atom, or an alkyloxy,alkyloxycarbonyl, amino, alkylamino or dialkylamino radical, orrepresent a benzyl or heterocyclyl radical, wherein said heterocyclylradical may be saturated or unsaturated and contains 5 or 6 ring membersand from 1 to 3 heteroatoms; or in which R₁₂ and R₁₃ form, together withthe nitrogen atom to which they are attached, a saturated or unsaturatedheterocycle containing 4 to 6 ring members, which heterocycle mayoptionally contain an additional heteroatom selected from nitrogen,oxygen and sulphur, and wherein said saturated or unsaturatedheterocycle is optionally substituted by an alkyl, phenyl or benzylradical, or

a radical of the formula —N(R₁₄)—(CH₂)_(n)—NR₁₂R₁₃ in which R₁₂ and R₁₃are as defined above R.₁₄ represents a hydrogen atom or an alkylradical, and n is an integer from 2 to 4;

wherein the alkyl portions or radicals defined above are straight chainor branched and contain from 1 to 4 carbon atoms;

or a pharmaceutically acceptable salt thereof.

15. The method of 14, wherein:

Alk represents a C₃-C₆ straight chain or branched alkylene radical, and

R represents

a hydroxyl radical, or

an —NR₁₂R₁₃ radical in which R₁₂ and R₁₃, which are identical ordifferent, represent a hydrogen atom or an alkyl, C₃₋₄ alkenyl oroptionally substituted phenyl radical, wherein said phenyl radical maybe substituted by a halogen atom or an alkyloxy, alkyloxycarbonyl,amino, alkylamino or dialkylamino radical, or represents a benzylradical; or in which R₁₂ and R₁₃ form, together with the nitrogen atomto which they are attached, a saturated or unsaturated heterocyclecontaining 4 to 6 ring members, which heterocycle may optionally containan additional heteroatom selected from nitrogen, oxygen and sulphur, andwherein said saturated or unsaturated heterocycle is optionallysubstituted by an alkyl radical;

or a pharmaceutically acceptable salt thereof.

16. The method of 14, wherein:

Alk represents a C₂₋₆ straight chain or branched alkylene radical, and

R represents a hydroxyl radical, or an —NR₁₂R₁₃ radical in which R₁₂ andR₁₃, which are identical or different, represent a hydrogen atom or analkyl, allyl, phenyl or benzyl radical; or in which R₁₂ and R₁₃ form,together with the nitrogen atom to which they are attached, aheterocycle selected from azetidinyl, piperidyl, piperazinyl,N-methylpiperazinyl, N-phenylpiperazinyl, N-benzylpiperazinyl,morpholino, tetrahydropyridyl, methyltetrahydropyridyl andphenyltetrahydropyridyl;

or a pharmaceutically acceptable salt thereof.

17. The method of 16, wherein the cyclosporine A derivative is selectedfrom the group consisting of[(R)-2-(N,N-dimethyl-amino)ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporinA,

-   [(R)-2-(1-piperidyl)-ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin    A,-   [(R)-2-(N-methyl-N-t-butylamino)ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin    A,-   [(R)-2-(hydroxy)-ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin A,-   [(R)-2-(N,N-diethyl-amino)ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin    A,-   [(R)-2-(N,N-dimethylamino)ethylthio-Sar]³-cyclosporin A,-   [(R)-2-(1-piperidyl)ethylthio-Sar]³-cyclosporin A,-   [(R)-2-(N-methyl-N-1-propylamino)ethylthio-Sar]³-cyclosporin A and-   [(R)-2-(N-methyl-N-t-butylamino)ethylthio-Sar]³-cyclosporin A, and    pharmaceutically acceptable salts thereof.

18. The method of 16, wherein the cyclosporine A derivative is(R)-(diethylaminoethylthio-Sar)³ cyclosporin A,

19. A method of treating inflammatory diseases selected from the groupconsisting of ocular inflammatory diseases, dermal inflammatorydiseases, inflammatory rheumatic diseases, inflammatory bowel disease,neuroinflammatory diseases and autoimmune hematological diseases anddisorders wherein said cyclosporine A derivative is selected from thegroup consisting of compounds represented by the formula:

wherein R₁ is S-Alk-R wherein Alk is an alkylene or alkylenyl linkage, Ris

—N═C(NR₃R₄)(NR₅R₆) or —NR₇[(NR₃R₄)C═NR₅], or —N═C(R₈)(NR₉R₁₀), whereinR₃-R₇ ₁₀ is H, Alk, Ar or (CH₂)nAr wherein Ar is an aryl group and n isan integer of from 1 to 13 or R₃ and R₄, or R₄ and R₅, or R₅ and R₇, orR₃ and R₇, or R₉ and R₁₀, or R₈ and R₉, together, may be —(CH₂)_(x)—,wherein x is an integer of from 2 to 5 and R₂ is selected from the groupconsisting of hydroxyl, lower alkyl and hydroxyl-substituted loweralkyl.

20. The method of 19 wherein R₁ is a methylene or a C₂ to C₆polymethylene linkage.

21. The method of 19 wherein R₁ is a C₃ to C₆ alkenylenyl linkage.

22. The method of 19 wherein R is —N═C(NR₃R₄)(NR₅R₆) or—NR₇C(NR₃)(C═NR₅), wherein R₃-R₇ is H, Alk, Ar or (CH₂)nAr wherein Ar isan aryl group and n is an integer of from 1 to 13 or R₃ and R₄ or R₄ andR₅ or R₅ and R₇ or R₃ and R₇, together may be —CH₂—CH₂— or—CH₂—CH₂—CH₂—.

23. The method of 19 wherein said cyclosporine A derivative is a3-substituted diaminoiminoalkylthio cyclosporine A derivative.

24. The method of 19 wherein said cyclosporine A derivative is selectedfrom the group consisting of((R)-(diamino)iminoalkyllthio-Sar)³-(4′-hydroxy-MeLeu)⁴ cyclosporin Aderivatives,((R)-(alkyl)(dialkylamino)iminoalkylthio-Sar)³-(4′-hydroxy-MeLeu)⁴-cyclosporinA, ((R)-(alkyl)(dialkylamino)iminoalkylthio-Sar)³-cyclosporin Aderivatives and ((R)-(diamino)iminoalkylthio-Sar)³-cyclosporin Aderivatives

25. The method of 19 wherein R₁ is a hydrogen atom or a radical offormula (Ia):

—S-Alk-R₁  (Ia)

in which

Alk-R₁₁ represents a methyl radical, or alternatively

Alk represents a C₂-C₆ straight chain or branched alkylene radical or aC₃-C₆ cycloalkylene radical, and

R₁₁. represents

a hydrogen atom or a hydroxyl, carboxyl or alkyloxycarbonyl radical, or

an —NR₁₂R₁₃ radical in which R₁₂ and R₁₃, which are identical ordifferent, represent a hydrogen atom or a phenyl, alkyl, C₂-C₄ alkenylor C₃-C₆ cycloalkyl radical, said radical optionally substituted withselected from a halogen atom, an alkyloxy, alkyloxycarbonyl, amino,alkylamino and dialkylamino radical; or

R₁₂ and R₁₃ represent a benzyl or saturated or unsaturated heterocycylicradical, said heterocycylic radical containing from 5 to 6 ring membersand from 1 to 3 heteroatoms;

or in which R₁₂ and R₁₃ form, together with the nitrogen atom to whichthey are attached, a saturated or unsaturated 4- to 6-memberedheterocycle, which heterocycle having an additional heteroatom selectedfrom nitrogen, oxygen and sulphur, and wherein said saturated orunsaturated heterocycle is optionally substituted by an alkyl, phenyl orbenzyl radical, or R₁ is a radical of the formula (Ib):—N(R₁₄)—(CH₂)_(n)—NR₁₂R₁₃ in which R₁₂ and R₁₃ are as defined above, R₁₄represents a hydrogen atom or an alkyl radical and n is an integerranging from 2 to 4,

and R₂ is selected from the group consisting of hydroxyl, lower alkyland hydroxyl substituted lower alkyl,

with the proviso that, when R₁ is a hydrogen atom, then R₂ is not analkyl butyl? radical, and wherein the alkyl portions or radicals definedabove are straight chain or branched and contain from 1 to 4 carbonatoms, or a pharmaceutically acceptable salt thereof.

26. The method of 25 wherein in the cyclosporine A derivatives of theformula, the trans butene moiety, which is normally present in the1-position of cyclosporine A, is replaced with R₁₅ wherein R₁₅represents a radical of formula

—CH₂CHCHCH₂—R₁₆ (Ic) or —CH₂SR₁₇ (Id), wherein R₁₆ represents analkylthio, aminoalkylthio, alkylaminoalkylthio, dialkylaminoalkylthio,pyrimidinylthio, thiazolylthio, N-alkylimidazolylthio,hydroxyalkylphenylthio, hydroxyalkylphenyloxy, nitrophenylamino or2-oxopyrimidin-1-yl radical and R₁₇ represents an alkyl radical.

27. The method of 25 wherein R represents

a hydroxyl radical,

a carboxyl radical,

an alkyloxycarbonyl radical,

an —NR₁₂R₁₃ radical in which R₁₂ and R₁₃, which are identical ordifferent, represent a hydrogen atom or an alkyl, C₂-C₄ alkenyl, C₃-C₆cycloalkyl or optionally substituted phenyl radical, wherein said phenylradical may be substituted by a halogen atom, or an alkyloxy,alkyloxycarbonyl, amino, alkylamino or dialkylamino radical, orrepresent a benzyl or heterocyclyl radical, wherein said heterocyclylradical may be saturated or unsaturated and contains 5 or 6 ring membersand from 1 to 3 heteroatoms; or in which R₁₂ and R₁₃ form, together withthe nitrogen atom to which they are attached, a saturated or unsaturatedheterocycle containing 4 to 6 ring members, which heterocycle mayoptionally contain an additional heteroatom selected from nitrogen,oxygen and sulphur, and wherein said saturated or unsaturatedheterocycle is optionally substituted by an alkyl, phenyl or benzylradical, or

a radical of the formula —N(R₁₄)—(CH₂)_(n)—NR₁₂R₁₃ in which R₁₂ and R₁₃are as defined above R.₁₄ represents a hydrogen atom or an alkylradical, and n is an integer from 2 to 4;

wherein the alkyl portions or radicals defined above are straight chainor branched and contain from 1 to 4 carbon atoms;

or a pharmaceutically acceptable salt thereof.

28. The method of 27, wherein:

Alk represents a C₃-C₆ straight chain or branched alkylene radical, and

R represents a hydroxyl radical, or an —NR₁₂R₁₃ radical in which R₁₂ andR₁₃, which are identical or different, represent a hydrogen atom or analkyl, C₃₋₄ alkenyl or optionally substituted phenyl radical, whereinsaid phenyl radical may be substituted by a halogen atom or an alkyloxy,alkyloxycarbonyl, amino, alkylamino or dialkylamino radical, orrepresents a benzyl radical; or in which R₁₂ and R₁₃ form, together withthe nitrogen atom to which they are attached, a saturated or unsaturatedheterocycle containing 4 to 6 ring members, which heterocycle mayoptionally contain an additional heteroatom selected from nitrogen,oxygen and sulphur, and wherein said saturated or unsaturatedheterocycle is optionally substituted by an alkyl radical;

or a pharmaceutically acceptable salt thereof.

29. The method of 27, wherein:

Alk represents a C₂₋₆ straight chain or branched alkylene radical, and

R represents a hydroxyl radical, or an —NR₁₂R₁₃ radical in which R₁₂ andR₁₃, which are identical or different, represent a hydrogen atom or analkyl, allyl, phenyl or benzyl radical; or in which R₁₂ and R₁₃ form,together with the nitrogen atom to which they are attached, aheterocycle selected from azetidinyl, piperidyl, piperazinyl,N-methylpiperazinyl, N-phenylpiperazinyl, N-benzylpiperazinyl,morpholino, tetrahydropyridyl, methyltetrahydropyridyl andphenyltetrahydropyridyl;

or a pharmaceutically acceptable salt thereof.

30. The method of 29, wherein the cyclosporine A derivative is selectedfrom the group consisting of

-   [(R)-2-(N,N-dimethyl-amino)ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin    A,-   [(R)-2-(1-piperidyl)-ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin    A,-   [(R)-2-(N-methyl-N-t-butylamino)ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin    A,-   [(R)-2-(hydroxy)-ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin A,-   [(R)-2-(N,N-diethyl-amino)ethylthio-Sar]³-[4′-hydroxy-MeLeu]⁴-cyclosporin    A,-   [(R)-2-(N,N-dimethylamino)ethylthio-Sar]³-cyclosporin A,-   [(R)-2-(1-piperidyl)ethylthio-Sar]³-cyclosporin A,-   [(R)-2-(N-methyl-N-1-propylamino)ethylthio-Sar]³-cyclosporin A and-   [(R)-2-(N-methyl-N-t-butylamino)ethylthio-Sar]³-cyclosporin A, and    pharmaceutically acceptable salts thereof.

31. The method of 30, wherein the cyclosporine A derivative is(R)-(diethylaminoethylthio-Sar)³ cyclosporin A,

32. The method of 19 wherein said ocular inflammatory disease iskeratoconjunctivitis sicca, vernal keratoconjunctivitis, allergicconjunctivitis, or uveitis

33. The method of 19 wherein said dermal inflammatory disease ispsoriasis or atopic dermatitis),

34. The method of 19 wherein said inflammatory rheumatic disease isrheumatoid arthritis, scleroderma, systemic lupus erythematosus, Wegenergranulamatosis, polymyositis, dermatomyositis, psoriatic arthritis,ankylosing spondylitis, Reiter's syndrome or juvenile rheumatoidarthritis),

35. The method of 19 wherein said ocular inflammatory bowel disease isulcerative colitis or Crohn's disease.

36. The method of 19 wherein said neuroinflammatory disease is multiplesclerosis.

37. The method of 19 wherein said ocular autoimmune hematologicaldisorder is hemolytic anaemia, aplastic anaemia, pure red cell anaemia,or idiopathic thrombocytopaenia.

What is claimed is:
 1. A method for the treatment of an inflammatorycondition of a mammal, comprising the step of administering to a patientin need thereof, a therapeutically effective amount of a cyclosporin Aderivative represented by the following formula:

wherein R₁ is S-Alk-R wherein Alk is an alkylene linkage and R is ahydrogen or a unsubstituted or substituted hydrocarbyl group.